Method for detecting biomaterial using linear upconversion fluorescent property

ABSTRACT

A method for detecting biomaterial by means of a dye having a linear upconversion fluorescent property is provided. The method includes the steps of: i) preparing a fluorophore having a linear upconversion fluorescent property; ii) reacting the fluorophore and biomaterial to obtain a reaction complex thereof; iii) exciting the reaction complex by means of a light source having a longer wavelength than the maximum light-emitting wavelength of the fluorophore; and iv) detecting and measuring the light-emitting signal having a shorter wavelength than the wavelength of the excited light emitted from the excited reaction complex. A system and a kit for detecting biomaterial using a dye having a linear upconversion fluorescent property are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Section 371 of International Application No.PCT/KR2016/005166, filed May 16, 2016, which was published in the Koreanlanguage on Nov. 24, 2016, under International Publication No. WO2016/186412 A1, which claims priority under 35 U.S.C. § 119(b) to KoreanPatent Application No. 10-2015-0067872, filed May 15, 2015, and KoreanPatent Application No. 10-2016-0059525, filed May 16, 2016, thedisclosures of which are incorporated herein by reference in theirentirety.

BACKGROUND OF THE INVENTION

The present invention relates to an assay method for detecting abiomaterial using a linear upconversion fluorescent property.

A target specimen (blood, sputum, etc.) or a detection substratebasically has fluorophores, which act as background noise in regards tofluorescence-based detection. In addition, when generally usedconventional fluorescent particles are used, such background noise isalso detected, and therefore, detection sensitivity of a target signalis limited.

One of the ways to minimize background noise is to utilize upconversionfluorescent materials, instead of general downconversion fluorescenceones, for a signaling label. Conventional upconversion nanoparticles(UCNPs) may have a size-independent maximum emission wavelength andfacilitate multiple color emissions by transforming a host crystal andan RE doping material. Taking advantage of such characteristics, UCNPshave been used in flow cytometry, photodynamic therapy, diagnosis, etc.,used as fluorescent labels for biological assays such as immunoassaysand gene analysis, and also used in chemical detection/cell imaging.

However, in order to detect fluorescence using conventional UCNPs, anon-linear upconversion type of inorganic nanocrystal (absorbs biphotonsand multiphotons) was mostly utilized, and a non-linear upconversiontype of inorganic nanocrystal is detected with a laser, which is ahigh-power coherent excitation light source and is expensive, whereindetection is difficult.

For that reason, the inventors developed novel linear UCNPs that can bedetected with a conventional LED by applying a linear upconversionfluorescent property to a method for detecting a biomaterial, and thusthe present invention was completed.

BRIEF SUMMARY OF THE INVENTION Disclosure Technical Problem

The present invention is directed to providing a method for detectingbiomaterial by means of a dye having a linear upconversion fluorescentproperty.

The present invention is also directed to providing a system fordetecting a biomaterial by means of a dye having a linear upconversionfluorescent property.

The present invention is also directed to providing a linearupconversion-based kit for diagnosing biomaterial, which includes afluorophore having a linear upconversion fluorescent property.

Technical Solution

To achieve the above-mentioned objects, the present invention provides amethod for detecting a biomaterial by means of a dye having a linearupconversion fluorescent property, the method including:

i) preparing a fluorophore having a linear upconversion fluorescentproperty;

ii) reacting the fluorophore with the biomaterial to form a reactioncomplex thereof;

iii) exciting the reaction complex using a light source with awavelength longer than the maximum emission wavelength of thefluorophore; and

iv) detecting and measuring a fluorescence signal with a wavelengthshorter than the wavelength of excitation light emitted from the excitedreaction complex.

The term “upconversion fluorescence” refers to a wide range of opticalphenomena in which high energy light is emitted by absorbing low energylight.

The term “linear upconversion fluorescence” used herein refers to alinear optical phenomenon based on the absorption of single photons, notthe absorption of multiple photons. Conventional non-linear upconversionfluorescence is a phenomenon of emitting high energy light bysequentially absorbing two or more photons from fluorescence, and thushas a difference from the linear upconversion of the present invention.

In the detection method of the present invention, there is no limitrelated to a fluorophore having a linear upconversion fluorescentproperty, and preferably, a fluorophore having a Stokes' shift of <100nm. The term “Stokes' shift” refers to the difference in excitationlight energy and light emission energy according to Strokes' Law.

In the detection method of the present invention, the fluorophore havinga linear upconversion fluorescent property may be, but is not limitedto, any one or more selected from the group consisting of a diketopyrrolo pyrrole (DPP) derivative represented by Formula 1 below, acene,fluorescein, rhodamine, oxazine, thiazine, cyanine, rubrene,boron-dipyrromethene (BODIPY), resorufin and hemicyanine. However, thefluorophore is not necessarily limited to the above examples, andtherefore may be any fluorophore having a linear upconversionfluorescent property, and preferably any fluorophore having a Stokes'shift of <100 nm.

(In Formula 1, R¹ and R² are the same or different, and are hydrogen, asubstituted or unsubstituted alkyl group having 1 to 60 carbon atoms, asubstituted or unsubstituted cycloalkyl group having 3 to 30 carbonatoms, a substituted or unsubstituted aryl group having 6 to 60 carbonatoms, a substituted or unsubstituted heteroaryl group having 2 to 60carbon atoms, or a substituted or unsubstituted condensed polycyclicgroup having 6 to 60 carbon atoms.)

In an exemplary embodiment of the present invention, the DPP derivativemay be, but is not limited to,2,5-bis(2-ethylhexyl)-3,6-di(thiophen-2-yl)pyrrolo[3,4-c]pyrrole-1,4(2H,5H)-dione[Formula 2];

2,5-bis(2-ethylhexyl)-2,5-dihydro-3,6-diphenyl-pyrrolo[3,4-c]pyrrole-1,4-dione[Formula 3];

5-(2,5-bis(2-ethylhexyl)-1,2,4,5-tetrahydro-1,4-dioxo-3-(thiophen-2-yl)pyrrolo[3,4-c]pyrrol-6-yl)thiophene-2-carbaldehyde[Formula 4]; or

5,5′-(2,5-bis(2-ethylhexyl)-3,6-dioxo-2,3,5,6-tetrahydropyrrolo[3,4-c]pyrrole-1,4-diyl)bis(thiophene-2-carbaldehyde)[Formula 5], and most preferably,2,5-bis(2-ethylhexyl)-3,6-di(thiophen-2-yl)pyrrolo[3,4-c]pyrrole-1,4(2H,5H)-dionerepresented by Formula 2.

In Step i) of the detection method of the present invention, thefluorophore may be directly conjugated to a molecule for detecting abiomaterial. Alternatively, the fluorophore is preferably loaded on orconjugated to a nanostructure, thereby forming a fluorescentnanostructure.

The term “nanostructure” used herein refers to a nano-scale structure,and here, the size of the nanostructure is not limited, and thenanostructure includes particles larger than nanoparticles, that is,microparticles. The nanostructure may have any size suitable for loadingor conjugating a fluorescent dye having a linear upconversionfluorescent property of the present invention.

The nanostructure may be, but is not limited to, any one or moreselected from the group consisting of nanoparticles, nanobeads, ananoemulsion, micelles, liposomes and a nanoparticle suspension. In anexemplary embodiment of the present invention, the nanostructure ispreferably polystyrene-based beads. In the present invention, thenanobeads may have a size of 1 to 1000 nm, but the size is not limitedthereto.

In Step ii) of the detection method of the present invention, thefluorophore may be conjugated to a molecule for detecting a biomaterial,and the molecule for detecting a biomaterial-conjugated fluorophore mayreact with the biomaterial, thereby forming a reaction complex thereof.

Here, the molecule for detecting a biomaterial may be any one or moreselected from the group consisting of a protein, an antibody, a gene, alipid, an enzyme, an aptamer and a ligand, but the present invention isnot limited thereto, and therefore any material capable ofcomplementarily binding to a biomaterial to be detected can be used.

In Step iii) of the detection method of the present invention, thereaction complex may be excited using a light source with a wavelength 5to 50 nm longer than the maximum emission wavelength of the fluorophore.Here, the light source may be, but is not limited to, a light emittingdiode (LED) or a lamp. That is, the light source of the presentinvention may be any light source other than a laser, and detection canbe executed with a conventional LED, which can give a cost reducingeffect when utilizing a fluorescence-measuring system of the presentinvention.

In Step iv) of the detection method of the present invention, anemission signal emitted from the reaction complex may be an emissionsignal in a wavelength range 10 to 100 nm shorter than a wavelength ofthe excitation light.

In the detection method of the present invention, the biomaterial maybe, but is not limited to, any one selected from the group consisting ofa tissue extract, a cell lysate, whole blood, plasma, serum, saliva,ocular humor, cerebrospinal fluid, sweat, urine, milk, ascitic fluid,sinovial fluid, peritoneal fluid and a dried blood spot.

The term “sample” used herein may be any sample capable of being appliedto a method or diagnostic kit of the present invention withoutlimitation, and particularly a liquid sample capable of being uniformlyapplied to a diagnostic kit. In the present invention, an analysissample may include all substances which can contain a targetbiomolecule, and particularly, various substances exposed within aliving body or isolated from the living body. The substances isolatedfrom the living body include, preferably, blood, urine, nasal mucus,cells, extracted DNA, RNA and a protein.

In addition, the detection method of the present invention may furtherinclude manufacturing a biosensor, biochip or kit for detecting abiomaterial by placing the molecule for detecting abiomaterial-conjugated linear upconversion fluorescent nanostructure ona biochip or kit for detecting a biomaterial or a strip sensor.

The present invention also relates to a biomaterial detection system 10,and referring to FIG. 1, the system 10 for detecting a biomaterial bymeans of a dye having a linear upconversion fluorescent property, whichincludes

a biomaterial diagnostic kit 120 consisting of an injection unit intowhich a biomaterial is injected, and a reaction unit which includes afluorophore having a linear upconversion fluorescent property and formsa reaction complex of the biomaterial and the fluorophore;

a light source unit 110 providing a light source to the reaction unit ofthe biomaterial diagnostic kit;

an emission signal collection unit 130 detecting an emission signal witha wavelength shorter than that of excitation light exciting thefluorophore of the reaction complex; and

a measurement unit 140 detecting and measuring the collected emissionsignal, wherein the light source unit 110 provides a light source with awavelength longer than the maximum emission wavelength of thefluorophore.

In the detection system, the fluorophore may be a fluorophore having aStokes' shift of <100 nm, but the present invention is not limitedthereto.

Meanwhile, the light source unit may include an excitation filter whichfiltrates light emitted from the light source, and the emission signalcollection unit may include a lens projecting the filtered light.However, the excitation filter is used so that only a predeterminedwavelength of the light source is able to penetrate. In addition, thelens of the emission signal collection unit may refer to an objectivelens and an eyepiece, and may include an emission filter between theobjective lens and the eyepiece like a common microscope. The objectivelens is used to concentrate radial light, and the emission filterfacilitates the measurement of light concentrated by the objective lensafter being delivered to the measurement unit (FIG. 1).

Such a light source unit, the filter and the lens may be arranged atsuitable intervals to easily observe the biomaterial.

In addition, in the detection system of the present invention, thediagnostic kit may be a rapid diagnostic kit or ELISA kit, but thepresent invention is not limited thereto.

Meanwhile, in an exemplary embodiment of the present invention, as thediagnostic kit, a rapid diagnostic kit may be used.

In this case, the rapid diagnostic kit may consist of a sample pad, aconjugate pad, a reaction unit consisting of a signal band and aconfirmation band, a membrane and an absorption pad. The sample pad mayrefer to an injecting unit for injecting a sample, and the conjugate padand the membrane may refer to the reaction unit.

More specifically, when a biomaterial is added to the sample pad, aliquid sample wets a dried sample pad, and is then transferred to theconjugate pad, and the analyte binds to an antibody stored in a driedstate, thereby forming a complex, followed by migration of the complexin the membrane. The signal band and the confirmation band are printedon the surface of the separation membrane, and when the analyte iscontained in the sample, the complex may be accumulated in the signalband region. When a certain amount or more of the complex isagglomerated by binding a substance such as gold particles or latexbeads to the antibody of the conjugate pad, the complex may be observedas a signal generated from the signal band unit.

In addition, since an antibody binding to the antibody of the storingunit is printed on the confirmation band of the membrane, the absence orpresence of the confirmation band is used as a criterion for determiningthe effectiveness of a test as a result of judging migration of theliquid sample to a part where necessary and action of the antibody, andthe absorption pad attached to an end of the separation membrane mayserve as pump by absorbing the liquid sample migrated through theseparation membrane to allow the liquid sample to continuously migrate.

More specifically, the biomaterial diagnostic kit of the presentinvention is used to perform a test by storing a fluorophore-chemicallylinked antibody in the conjugate pad in a dried state as a result ofutilizing a linear upconversion fluorescent property, and injecting thesample (biomaterial) into the sample pad. Afterward, an analyte in thesample (biomaterial), which has migrated into the conjugate pad, forms aconjugate with the stored antibody, and then the conjugate moves to theseparation membrane. The conjugate is accumulated in the signal band,and a remaining fluorophore-binding antibody is absorbed by theabsorption pad through the separation membrane. Here, the conjugatebetween the analyte selectively adsorbed by the signal band and thefluorophore-linked antibody may produce fluorescence by receiving energyfrom the light source unit.

In addition, in the detection system, the fluorophore having a linearupconversion fluorescent property may be, but is not limited to, any oneor more selected from the group consisting of a diketo pyrrolo pyrrole(DPP) derivative represented by Formula 1 below, acene, fluorescein,rhodamine, oxazine, thiazine, cyanine, rubrene, boron-dipyrromethene(BODIPY), resorufin and hemicyanine, and may be any fluorophore having alinear upconversion fluorescent property, and preferably any fluorophorehaving a Stokes' shift of <100 nm.

(In Formula 1, R¹ and R² are the same or different, and are hydrogen, asubstituted or unsubstituted alkyl group having 1 to 60 carbon atoms, asubstituted or unsubstituted cycloalkyl group having 3 to 30 carbonatoms, a substituted or unsubstituted aryl group having 6 to 60 carbonatoms, a substituted or unsubstituted heteroaryl group having 2 to 60carbon atoms, or a substituted or unsubstituted condensed polycyclicgroup having 6 to 60 carbon atoms.)

In an exemplary embodiment of the present invention, the DPP derivativemay be, but is not limited to,2,5-bis(2-ethylhexyl)-3,6-di(thiophen-2-yl)pyrrolo[3,4-c]pyrrole-1,4(2H,5H)-dione;2,5-bis(2-ethylhexyl)-2,5-dihydro-3,6-diphenyl-pyrrolo[3,4-c]pyrrole-1,4-dione;5-(2,5-bis(2-ethylhexyl)-1,2,4,5-tetrahydro-1,4-dioxo-3-(thiophen-2-yl)pyrrolo[3,4-c]pyrrol-6-yl)thiophene-2-carbaldehyde;or5,5′-(2,5-bis(2-ethylhexyl)-3,6-dioxo-2,3,5,6-tetrahydropyrrolo[3,4-c]pyrrole-1,4-diyl)bis(thiophene-2-carbaldehyde),and most preferably,2,5-bis(2-ethylhexyl)-3,6-di(thiophen-2-yl)pyrrolo[3,4-c]pyrrole-1,4(2H,5H)-dionerepresented by Formula 2.

In the detection system of the present invention, the fluorophore may bedirectly conjugated to a molecule for detecting a biomaterial. Inaddition, the fluorophore may be loaded on or conjugated to ananostructure, thereby forming a fluorescent nanostructure.

The nanostructure may be, but is not limited to, any one selected fromthe group consisting of nanoparticles, nanobeads, a nanoemulsion,micelles, liposomes and a nanoparticle suspension. In an exemplaryembodiment of the present invention, the nanostructure is preferablypolystyrene-based beads. In the present invention, the nanobeads mayhave a size of 1 to 1000 nm, but the size is not limited thereto.

The reaction complex of the biomaterial and the fluorophore of thedetection system may be formed by conjugating the fluorophore to themolecule for detecting a biomaterial in the reaction unit, and reactingthe molecule for detecting a biomaterial-conjugated fluorophore with thebiomaterial, but the present invention is not limited thereto.

In the detection system, the molecule for detecting a biomaterial may beany one or more selected from the group consisting of a protein, anantibody, a gene, a lipid, an enzyme, an aptamer and a ligand, but thepresent invention is not limited thereto, and therefore any materialcapable of complementarily binding to a biomaterial to be detected canbe used.

In the detection system, the light source with a wavelength longer thanthe maximum emission wavelength of the fluorophore may be, but is notlimited to, a light source with a wavelength 5 to 50 nm longer than themaximum emission wavelength of the fluorophore. Here, the light sourcemay be, but is not limited to, a light emitting diode (LED) or a lamp.That is, the light source of the present invention may be any lightsource other than a laser, and detection can be executed with aconventional LED, which can give a cost reducing effect when utilizing afluorescence-measuring system of the present invention.

In the detection system, the emission signal of the fluorophore may bean emission signal with a wavelength shorter than a wavelength of theexcitation light, and may be an emission signal in a wavelength range 10to 100 nm shorter than a wavelength of the excitation light.

In the detection system, the biomaterial may be, but is not limited to,any one selected from the group consisting of a tissue extract, a celllysate, whole blood, plasma, serum, saliva, ocular humor, cerebrospinalfluid, sweat, urine, milk, ascitic fluid, sinovial fluid, peritonealfluid and a dried blood spot.

In addition, the present invention provides a linear upconversion-basedbiomaterial diagnostic kit, which includes a fluorophore having a linearupconversion fluorescent property.

The term “diagnostic kit” used herein may include the linearupconversion fluorescent nanoparticles of the present invention and abiomaterial-detecting molecule, and further include other reagents,tools, etc., necessary for a process of detecting a target biomaterial.In addition, the diagnostic kit may be manufactured as a rapiddiagnostic kit capable of detecting the biomaterial of the presentinvention, or may be manufactured as other various types of diagnostickits.

The biomaterial-detecting molecule may be any one or more selected fromthe group consisting of a protein, an antibody, a gene, a lipid, anenzyme, an aptamer and a ligand, but the present invention is notlimited thereto, and most preferably an antibody.

The term “biomaterial” used herein refers to a material that can befound inside or outside of a living body, and includes all materialsgenerating a specific reaction in the living body, creating a specificresponse in the living body or produced by a specific state or reaction.Preferably, the biomaterial of the present invention is a compound, aprotein, DNA, RNA or cells. The term “target biomaterial” used hereinrefers to a target substance of interest of which the presence orcontent is detected using the diagnostic kit of the present invention,and specifically, a substance that specifically interacts with adetection molecule constituting the diagnostic kit.

The linear upconversion fluorescent nanostructure may be prepared byloading or conjugating a fluorescent dye having a linear upconversionfluorescent property on or to a nanostructure.

The fluorescent dye having a linear upconversion fluorescent propertymay be any one or more selected from the group consisting of a diketopyrrolo pyrrole (DPP) derivative represented by Formula 1 below, acene,fluorescein, rhodamine, oxazine, thiazine, cyanine, rubrene,boron-dipyrromethene (BODIPY), resorufin and hemicyanine.

(In Formula 1, R¹ and R² are the same or different, and are hydrogen, asubstituted or unsubstituted alkyl group having 1 to 60 carbon atoms, asubstituted or unsubstituted cycloalkyl group having 3 to 30 carbonatoms, a substituted or unsubstituted aryl group having 6 to 60 carbonatoms, a substituted or unsubstituted heteroaryl group having 2 to 60carbon atoms, or a substituted or unsubstituted condensed polycyclicgroup having 6 to 60 carbon atoms.)

In order to observe fluorescence using conventional UCNPs, a non-linearupconversion type of inorganic nanocrystal (absorbs biphotons andmultiphotons) was mostly utilized, and the non-linear upconversion typeof inorganic nanocrystal is observed with a laser, which is a high-powercoherent excitation light source and is expensive, wherein observationis difficult. However, the linear upconversion fluorescentnanoparticle-based diagnostic kit of the present invention can beobserved with a LED, which can give a cost reducing effect.

In an exemplary embodiment of the present invention,3,6-di(thiophen-2-yl)pyrrolo[3,4-c]pyrrole-1,4(2H,5H)-dione wassynthesized using thiophene-2 carbonitrile and dimethyl succinate, andthen ethylhexyl bromide was added, thereby synthesizing2,5-bis(2-ethylhexyl)-3,6-di(thiophen-2-yl)pyrrolo[3,4-c]pyrrole-1,4(2H,5H)-dione.Afterward,2,5-bis(2-ethylhexyl)-3,6-di(thiophen-2-yl)pyrrolo[3,4-c]pyrrole-1,4(2H,5H)-dionewas loaded on polystyrene beads to synthesize novel linear UCNPs, and ithas been confirmed that the developed particles exhibit an upconversionfluorescent property. In addition, a diagnostic system for a rapid kitwas developed by means of the novel linear UCNPs of the presentinvention.

Unless defined otherwise, all the technical and scientific terms used inthe specification have the same meanings as conventionally understood bythose of ordinary skill in the art to which the present inventionbelongs. Generally, the nomenclature used herein is well known in theart and conventionally used.

Advantageous Effects

The linear upconversion fluorescent nanostructure of the presentinvention can detect only signals of particles without background noise,thereby dramatically enhancing an S/N ratio and sensitivity. Inaddition, since fluorescence of the linear upconversion nanostructure ofthe present invention can be observed even with a conventional LED, acost reducing effect can be imparted when utilizing afluorescence-measuring system of the present invention.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

The foregoing summary, as well as the following detailed description ofthe invention, will be better understood when read in conjunction withthe appended drawings. For the purpose of illustrating the invention,there are shown in the drawings embodiments which are presentlypreferred. It should be understood, however, that the invention is notlimited to the precise arrangements and instrumentalities shown.

In the drawings:

FIG. 1 is a block diagram of a biomaterial detection system of thepresent invention;

FIG. 2A shows absorption and fluorescence spectra of DPP-Th of thepresent invention and FIG. 2B shows linear upconversion fluorescencespectra of DPP-Th-loaded polystyrene nanobeads (DPP-Th:2,5-bis(2-ethylhexyl)-3,6-di(thiophen-2-yl)pyrrolo[3,4-c]pyrrole-1,4(2H,5H)-dione);

FIG. 3 shows a linear upconversion fluorescent property based on theconcentration of a loaded fluorophore of DPP-Th-loaded polystyrenenanobeads of the present invention (DPP-Th:2,5-bis(2-ethylhexyl)-3,6-di(thiophen-2-yl)pyrrolo[3,4-c]pyrrole-1,4(2H,5H)-dione);

FIG. 4 is a schematic diagram of a rapid kit for diagnosing abiomaterial prepared by means of a linear upconversion fluorophore ofthe present invention;

FIG. 5 is a schematic diagram illustrating the configuration of adetection system for the linear upconversion fluorescence-based rapidkit of the present invention;

FIG. 6 shows a measurement system for the rapid kit capable of detectinga biomaterial by means of linear UCNPs according to the presentinvention;

FIG. 7 shows the sensitivity of DPP-Th-loaded nanobeads towards a linearupconversion fluorescence signal, compared with a general downconversionfluorescence signal, on a diagnostic strip of the rapid kit of thepresent invention;

FIGS. 8A, 8B, 8C, and 8D show the results of comparing the sensitivityof linear upconversion fluorescence signal with the sensitivity ofdownconversion fluorescence signal of DPP-Th-loaded nanobeads (FIG. 8A),rubrene-loaded polymer surfactant micelle nanoparticles (micelle,Pluronic F127) (FIG. 8B), a resorufin solution (FIG. 8C) and a rhodamineB solution (FIG. 8D), based on human serum, on a diagnostic strip of therapid kit of the present invention;

FIGS. 9A and 9B show the results of identifying immune responses on arapid kit-based membrane by means of linear UCNPs according to thepresent invention;

FIG. 10 shows the result of application of a rapid diagnostic kit bymeans of linear UCNPs according to the present invention; and

FIGS. 11A and 11B show the result of gene analysis by utilizing a linearupconversion fluorescent property according to the present invention.

DETAILED DESCRIPTION OF THE INVENTION Modes of the Invention

Hereinafter, the present application will be described in further detailwith reference to examples. The examples are merely provided to morefully describe the present application, and it will be obvious to thoseof ordinary skill in the art that the scope of the present applicationis not limited to the following examples.

Example 1. Synthesis of Linear Upconversion Dye (DPP-Th) Example 1-1.Synthesis of 3,6-di(thiophen-2-yl)pyrrolo[3,4-c]pyrrole-1,4(2H,5H)-dione

A mixture of sodium (2.65 g, 0.13 mol) and FeCl₃ was dissolved in t-amylalcohol (250 mL). The mixture was stirred at 110° C. until it becamelight yellow. After heating for 3 hours at 110° C., thiophene-2carbonitrile (11.9 g, 0.12 mol) was added to the reaction mixture whichwas then maintained for 0.5 hour. After slowly adding dimethyl succinate(5.29 g, 0.04 mol) drop-wise for 1.5 hours, the reaction mixture wasstirred at 120° C. for 4 hours. Subsequently, following cooling to 0°C., the reaction mixture was precipitated with MeOH/HCl. A solidobtained thereby was purified, washed with hot MeOH, and dried undervacuum. Thus, 10.2 g of dark red solid3,6-di(thiophen-2-yl)pyrrolo[3,4-c]pyrrole-1,4(2H,5H)-dione (84.8%) wasobtained, and used without purification.

Example 1-2. Synthesis of2,5-bis(2-ethylhexyl)-3,6-di(thiophen-2-yl)pyrrolo[3,4-c]pyrrole-1,4(2H,5H)-dione(DPP-Th)

The 3,6-di(thiophen-2-yl)pyrrolo[3,4-c]pyrrole-1,4(2H,5H)-dione (4 g,0.013 mol) obtained in Example 1-1 and K₂CO₃ (5.5 g, 0.039 mol) weresuspended in DMF (140 mL) under an argon atmosphere. The reactionmixture was stirred at 110° C. for 3 hours. After adding 2-ethylhexylbromide (9 g, 0.046 mol) drop-wise, the reaction mixture was maintainedfor 24 hours. After cooling at room temperature, a solvent was removedusing a rotary evaporator. Subsequently, water was added to purify acrude product. A residue was recrystallized with MeOH and purified.Finally, 1.1 g of brown solid2,5-bis(2-ethylhexyl)-3,6-di(thiophen-2-yl)pyrrolo[3,4-c]pyrrole-1,4(2H,5H)-dione(15.7%) was obtained.

Example 2. Preparation of Linear Upconversion Nanobeads and FluorescentCharacteristic of Linear Upconversion Nanobeads

100 μl of a tetrahydrofuran (THF) solution containing 5×10⁻⁵ M DPP-Thobtained in Example 1 was added to 600 μl of 0.4 w/v % carboxylatedpolystyrene beads (100 nm, Invitrogen). A mixture solution was slowlystirred at room temperature for 30 minutes. Afterward, THF was removedwith a rotary evaporator, resulting in linear upconversion nanobeads.

In addition, FIG. 2A shows absorption and fluorescence spectra of DPP-Thof the present invention and FIG. 2B shows linear upconversionfluorescence spectra of DPP-Th-loaded polystyrene nanobeads (DPP-Th:2,5-bis(2-ethylhexyl)-3,6-di(thiophen-2-yl)pyrrolo[3,4-c]pyrrole-1,4(2H,5H)-dione).It can be confirmed that the fluorophore has a Stokes' shift ofapproximately 20 nm, and the loaded polystyrene nanobeads have afluorescent component in a wavelength range (540-570 nm) shorter thanthat of excitation light under excitation at 580 nm.

Specifically, a fluorescent property of the novel linear upconversionnanostructure was confirmed. As a result, it was confirmed that thedeveloped particles have an upconversion fluorescent component emittingshorter wavelength light as a result of absorbing longer wavelengthlight. In addition, fluorescence intensity based on the concentration ofthe loaded fluorescent dye was measured. The results are shown in FIG.3.

Example 3. Development of Linear UCNP-Based Rapid Diagnostic Kit andMeasurement System

A rapid kit for diagnosing a biomaterial by means of the linear UCNPs ofExample 2 was developed (FIG. 4).

The rapid kit 120 was manufactured based on a generally knownconfiguration. Specifically, a rapid diagnostic kit 120 including asample pad 121, a conjugation pad 122, a membrane 125 and an absorptionpad was manufactured.

Meanwhile, the sample pad of the rapid diagnostic kit 120 refers to aninjection unit 121 of the present invention, and the conjugation pad122, a signal pad 123, a confirmation band 124, the membrane 125 and anabsorption pad refer to a reaction unit 126.

In addition, a biomaterial detection system 10 shown in FIG. 5 wasconstructed by means of the linear UCNP-based rapid diagnostic kit 120.Specifically, a LED 110 was disposed such that excitation light wasapplied to the rapid diagnostic kit 120 through an excitation filter(not shown), and fluorescence generated from the diagnostic kit 120 wasdisposed so as to reach a measurement unit 140 through an objective lens131, an emission filter 132 and an eyepiece 133. The objective lens 131was arranged to adjust a focal distance on the kit 120, and designed tomeasure a fluorescence signal in a corresponding focal region on the kit120 (FIGS. 5 and 6).

Example 4. Test for Fluorescent Signal Sensitivity of LinearUpconversion Nanobeads on a Serum Sample

Linear upconversion fluorescence signal sensitivity was measured bymeans of the linear UCNPs of Example 2, compared to serum (background),on a diagnostic strip (FIG. 7). First, 1 μl of fetal bovine serum (FBS)and DPP-Th nanobeads (4 mg/ml in DW) diluted in FBS at 1/10 were placedon diagnostic strips, respectively. Afterward, a linear upconversionfluorescent property was confirmed using an imaging system according tothe conditions shown in Table 1 below. Linear upconversion fluorescentand general fluorescent properties mentioned herein are measured underthe conditions in Table 1 below.

TABLE 1 Conditions for measuring linear upconversion fluorescence andgeneral downconversion fluorescence General fluorescence Linearupconversion (downconversion) fluorescence Wavelength of excitation 500nm 590 nm light Measured emission 585 nm 550 nm wavelength

Referring to FIG. 7, it can be seen that a target signal with highsensitivity without background noise was measured using a light sourcesuch as an LED by utilizing a linear upconversion fluorescent propertyaccording to the present invention.

Example 5. Test for Fluorescence Signal Sensitivity of LinearUpconversion Fluorophore on a Serum Sample

Linear upconversion fluorescence signal sensitivity and downconversionfluorescence signal sensitivity of the DPP-Th-loaded nanobeads ofExample 2, rubrene-loaded polymer surfactant micelle nanoparticles(micelle; Pluronic F127), a resorufin aqueous solution, and a rhodamineB aqueous solution were compared based on a serum sample. Therubrene-loaded micelle nanoparticles were prepared by drying 20 mg of apolymer surfactant (Pluronic F127) and 1 mg of rubrene in a THFsolution, and adding distilled water for reconstitution (diluted to thefinal rubrene concentration of 4×10⁻⁴M). The resorufin aqueous solutionand the rhodamine B aqueous solution were prepared in aphosphate-buffered solution (PBS, pH 8.0) with distilled water at aconcentration of 10⁻⁵M, respectively, and DPP-Th was prepared by beingdiluted with a dye concentration of 10⁻⁵ M. The materials were mixedwith human serum at a volume ratio of 1:1 and placed on diagnosticstrips to identify linear upconversion fluorescent properties (FIGS.8A-8D). As shown in FIGS. 8A-8D, it was identified that the linearupconversion fluorescence according to the present invention has almostno background noise caused by the autofluorescence of serum as comparedto general fluorescence.

Example 6. Confirmation of Immune Response Using Linear UCNPs

A surface of the linear UCNPs of Example 2 was modified with antibodies,and immune response tests were carried out using serum and proteinsolutions on a nitrocellulose membrane. The antibody modificationprocess is as follows: an 1-ethyl-3-(3-dimethylaminopropyl)carbodiimidehydrochloride aqueous solution (30 μl, 100 mg/ml) was added to anaqueous solution containing the linear UCNPs of Example 2 (1 ml, 3mg/ml), and the resulting mixture was reacted at room temperature for 15minutes. Here, an anti C-reactive protein (CRP) polyclonal antibody(pAb-CRP; Millipore, Calbiochem) was added (15 μl, 10 mg/ml) and reactedat room temperature for 90 minutes. The resulting product wascentrifuged at 12,000 rpm for 15 minutes, washed three times, andblocked with 3% bovine serum albumin (BSA).

An experiment for confirming an immune response was performed on anitrocellulose membrane as follows. A membrane to which a controlantibody (antirabbit IgG; R&D Systems) and a test antibody (mAb-CRP; R&Dsystem) were fixed was blocked with a blocking buffer (1% BSA-containingPBS), and washed with PBS three times. Afterward, a solution in whichthe antibody-modified linear UCNPs, an antigen (CRP) and serum weremixed was applied to an entire surface of the membrane, and then washedwith PBST three times. Following the immune response experiment,fluorescence of the nitrocellulose membrane was measured, and theresults are shown in FIGS. 9A and 9B. As shown in FIG. 9A, adsorption ofserum itself to the entire surface of the membrane after PBST washingcan be confirmed by means of general fluorescence. It can be seen that,when the mixture of the antigen, the antibody-modified linear UCNPs andthe serum was applied, strong fluorescence was selectively emitted fromthe control and the test antibody-fixed region. From the generalfluorescence, a fluorescence signal based on the adsorption of the serumcontained in the solution applied by the membrane was also detected, andit acted as strong background noise, and therefore it can be seen that asignal of nanoparticles binding to the antibody-fixed region is measuredwith high sensitivity in the linear upconversion fluorescence.

Example 7. Application of Linear UCNP-Based Rapid Diagnostic Kit

The antibody-modified linear UCNPs of Example 6 were loaded onto theconjugation pad of the diagnostic kit according to Example 3, and then aparticle flow property caused by eluent loading was observed with alinear upconversion signal. The result is shown in FIG. 10. As shown inFIG. 10, the particle flow property was identified in a linearupconversion fluorescence mode, and a signal representing minimalbackground noise in the membrane region can be observed.

Example 8. Linear Upconversion Fluorescence-Based Gene Analysis

The linear upconversion fluorescent property was applied to geneanalysis. A method for detecting a target gene by means of probegene-conjugated metal nanoparticles has been well-known. In thisexample, a linear upconversion fluorescence technique according to thepresent invention was applied to the conventionally known method.Specifically, a gold nanoparticle-fluorophore platform (gold nanobeacon)may be manufactured to be utilized as a turn-on fluorescent sensorthrough target DNA hybridization. Gene sequences used in the geneanalysis are as follows:

Probe DNA sequence (hairpin type):5′-SH-(CH₂)₆-TCG CTG AGA TCG GGA TCC CCA AAA TCA GCG A-(CH₂)₆-TAMRA-3′(Bioneer) Target DNA sequence: 5′-GGG GAT CCC GAT CTC AG-3′ (Bioneer)

15 nm Au NP and probe DNA were reacted at a molar ratio of 1:100. Then,a NaCl solution (5 M) was added to the reaction product at one-hourintervals a total of four times to adjust the final NaCl concentrationto 0.3 M. In addition, the resulting product was purified through threecycles of centrifugation (14,000 rpm, 30 min, 4° C.).

The probe DNA-conjugated gold nanoparticles were reacted with targetDNA, and then fluorescence was measured. Specifically, 1.5 nM nanobeaconwas reacted with 100 nM target DNA (pH 7 Tris buffer) at 37° C. for 1hour, and generated fluorescence signals were compared depending on theabsence or presence of serum. The result is shown in FIGS. 11A-11B.

Referring to FIGS. 11A and 11B, fluorescence of an extinct fluorophore(TAMRA) dequenched by the target DNA can be observed in a serum-freeenvironment. It is observed in a general fluorescence mode, and thefluorescence signal generated by the target DNA is hidden by theautofluorescence of the serum in a serum-present environment (50% volumeratio). However, it can be seen that the fluorescence signal generatedfrom the fluorophore can be clearly observed regardless of the absenceor presence of serum in a linear upconversion fluorescence mode.

EXPLANATION OF REFERENCE NUMERALS

-   -   10: Biomaterial detection system    -   110: Light source unit    -   120: Diagnostic kit    -   121: Injection unit    -   122: Conjugate pad    -   123: Signal band    -   124: Confirmation band    -   125: Membrane    -   126: Reaction unit    -   130: Generated signal collection unit    -   131: Objective lens    -   132: Emission filter    -   133: Eyepiece    -   140: Measurement unit

It will be appreciated by those skilled in the art that changes could bemade to the embodiments described above without departing from the broadinventive concept thereof. It is understood, therefore, that thisinvention is not limited to the particular embodiments disclosed, but itis intended to cover modifications within the spirit and scope of thepresent invention as defined by the appended claims.

We claim:
 1. A method for detecting a biomaterial by means of a dyehaving a linear upconversion fluorescent property, the methodcomprising: i) preparing a fluorophore having a linear upconversionfluorescent property; ii) reacting the fluorophore with the biomaterialto form a reaction complex thereof; iii) exciting the reaction complexusing a light source with a wavelength longer than the maximum emissionwavelength of the fluorophore; and iv) detecting and measuring afluorescence signal with a wavelength shorter than the wavelength ofexcitation light emitted from the excited reaction complex.
 2. Themethod of claim 1, wherein the fluorophore has a Stokes' shift of <100nm.
 3. The method of claim 1, wherein the fluorophore having a linearupconversion fluorescent property is any one or more selected from thegroup consisting of a diketo pyrrolo pyrrole (DPP) derivativerepresented by Formula 1 below, acene, fluorescein, rhodamine, oxazine,thiazine, cyanine, rubrene, boron-dipyrromethene (BODIPY), resorufin andhemicyanine:

where R¹ and R² are the same or different, and are hydrogen, asubstituted or unsubstituted alkyl group having 1 to 60 carbon atoms, asubstituted or unsubstituted cycloalkyl group having 3 to 30 carbonatoms, a substituted or unsubstituted aryl group having 6 to 60 carbonatoms, a substituted or unsubstituted heteroaryl group having 2 to 60carbon atoms, or a substituted or unsubstituted condensed polycyclicgroup having 6 to 60 carbon atoms.
 4. The method of claim 3, wherein theDPP derivative is2,5-bis(2-ethylhexyl)-3,6-di(thiophen-2-yl)pyrrolo[3,4-c]pyrrole-1,4(2H,5H)-dione;2,5-bis(2-ethylhexyl)-2,5-dihydro-3,6-diphenyl-pyrrolo[3,4-c]pyrrole-1,4-dione;5-(2,5-bis(2-ethylhexyl)-1,2,4,5-tetrahydro-1,4-dioxo-3-(thiophen-2-yl)pyrrolo[3,4-c]pyrrol-6-yl)thiophene-2-carbaldehyde;or5,5′-(2,5-bis(2-ethylhexyl)-3,6-dioxo-2,3,5,6-tetrahydropyrrolo[3,4-c]pyrrole-1,4-diyl)bis(thiophene-2-carbaldehyde).5. The method of claim 1, wherein, in Step i), the fluorophore is loadedon or conjugated to a nanostructure, thereby forming a fluorescentnanostructure.
 6. The method of claim 5, wherein the nanostructure isany one or more selected from the group consisting of nanoparticles,nanobeads, a nanoemulsion, micelles, liposomes and a nanoparticlesuspension.
 7. The method of claim 1, wherein, in Step ii), thefluorophore is conjugated to a molecule for detecting a biomaterial, andthe molecule for detecting a biomaterial-conjugated fluorophore mayreact with the biomaterial, thereby forming a reaction complex thereof.8. The method of claim 7, wherein the molecule for detecting abiomaterial is any one or more selected from the group consisting of aprotein, an antibody, a gene, a lipid, an enzyme, an aptamer and aligand.
 9. The method of claim 1, wherein in Step iii), the reactioncomplex is excited using a light source with a wavelength 5 to 50 nmlonger than the maximum emission wavelength of the fluorophore.
 10. Themethod of claim 1, wherein the light source in Step iii) is a laser, alight emitting diode (LED) or a lamp.
 11. The method of claim 1,wherein, in Step iv), the emission signal emitted from the reactioncomplex is an emission signal in a wavelength range 10 to 100 nm shorterthan that of the excitation light.
 12. The method of claim 1, whereinthe biomaterial is selected from the group consisting of a tissueextract, a cell lysate, whole blood, plasma, serum, saliva, ocularhumor, cerebrospinal fluid, sweat, urine, milk, ascitic fluid, sinovialfluid, peritoneal fluid and a dried blood spot.
 13. A system fordetecting a biomaterial by means of a dye having a linear upconversionfluorescent property, comprising: a biomaterial diagnostic kitconsisting of an injection unit into which a biomaterial is injected,and a reaction unit which includes a fluorophore having a linearupconversion fluorescent property and forms a reaction complex of thebiomaterial and the fluorophore; a light source unit configured toprovide a light source to the reaction unit of the biomaterialdiagnostic kit; an emission signal collection unit configured to detectan emission signal with a wavelength shorter than that of excitationlight exciting the fluorophore of the reaction complex; and ameasurement unit configured to detect and measure the collected emissionsignal, wherein the light source unit provides a light source with awavelength longer than the maximum emission wavelength of thefluorophore.
 14. The system of claim 13, wherein the light source unitcomprises an excitation filter which filtrates light emitted from thelight source; and a lens located on a top surface of the excitationfilm, and configured to project the filtered light.
 15. The system ofclaim 13, wherein the fluorophore has a Stokes' shift of <100 nm. 16.The system of claim 13, wherein the fluorophore having a linearupconversion fluorescent property is any one or more selected from thegroup consisting of a diketo pyrrolo pyrrole (DPP) derivativerepresented by Formula 1 below, acene, fluorescein, rhodamine, oxazine,thiazine, cyanine, rubrene, boron-dipyrromethene (BODIPY), resorufin andhemicyanine:

where R¹ and R² are the same or different, and are hydrogen, asubstituted or unsubstituted alkyl group having 1 to 60 carbon atoms, asubstituted or unsubstituted cycloalkyl group having 3 to 30 carbonatoms, a substituted or unsubstituted aryl group having 6 to 60 carbonatoms, a substituted or unsubstituted heteroaryl group having 2 to 60carbon atoms, or a substituted or unsubstituted condensed polycyclicgroup having 6 to 60 carbon atoms.
 17. The system of claim 16, whereinthe DPP derivative is2,5-bis(2-ethylhexyl)-3,6-di(thiophen-2-yl)pyrrolo[3,4-c]pyrrole-1,4(2H,5H)-dione;2,5-bis(2-ethylhexyl)-2,5-dihydro-3,6-diphenyl-pyrrolo[3,4-c]pyrrole-1,4-dione;5-(2,5-bis(2-ethylhexyl)-1,2,4,5-tetrahydro-1,4-dioxo-3-(thiophen-2-yl)pyrrolo[3,4-c]pyrrol-6-yl)thiophene-2-carbaldehyde;or5,5′-(2,5-bis(2-ethylhexyl)-3,6-dioxo-2,3,5,6-tetrahydropyrrolo[3,4-c]pyrrole-1,4-diyl)bis(thiophene-2-carbaldehyde).18. The system of claim 13, wherein the fluorophore is loaded on orconjugated to a nanostructure, thereby forming a fluorescentnanostructure.
 19. The system of claim 18, wherein the nanostructure isany one selected from the group consisting of nanoparticles, nanobeads,a nanoemulsion, micelles, liposomes and a nanoparticle suspension. 20.The system of claim 13, wherein the reaction complex of the biomaterialand the fluorophore of the detection system is formed by conjugating thefluorophore to a molecule for detecting a biomaterial in the reactionunit, and reacting the molecule for detecting a biomaterial-conjugatedfluorophore with the biomaterial.
 21. The system of claim 20, whereinthe molecule for detecting a biomaterial is any one or more selectedfrom the group consisting of a protein, an antibody, a gene, a lipid, anenzyme, an aptamer and a ligand.
 22. The system of claim 13, wherein thelight source with a wavelength longer than the maximum emissionwavelength of the fluorophore is a light source with a wavelength range5 to 50 nm longer than the maximum emission wavelength of thefluorophore.
 23. The system of claim 13, wherein the light source of theexcitation light source unit is a light emitting diode (LED) or a lamp.24. The system of claim 13, wherein the emission signal of thefluorophore is an emission signal in a wavelength range 10 to 100 nmshorter than the excitation wavelength.
 25. The system of claim 13,wherein the biomaterial is selected from the group consisting of atissue extract, a cell lysate, whole blood, plasma, serum, saliva,ocular humor, cerebrospinal fluid, sweat, urine, milk, ascitic fluid,sinovial fluid, peritoneal fluid and a dried blood spot.
 26. The systemof claim 13, wherein the diagnostic kit is a rapid diagnostic kit orELISA kit.
 27. A linear upconversion-based biomaterial diagnostic kit,comprising a fluorophore having a linear upconversion fluorescentproperty.